NAD+ repletion with niacin counteracts cancer cachexia

Cachexia is a debilitating wasting syndrome and highly prevalent comorbidity in cancer patients. It manifests especially with energy and mitochondrial metabolism aberrations that promote tissue wasting. We recently identified nicotinamide adenine dinucleotide (NAD+) loss to associate with muscle mitochondrial dysfunction in cancer hosts. In this study we confirm that depletion of NAD+ and downregulation of Nrk2, an NAD+ biosynthetic enzyme, are common features of severe cachexia in different mouse models. Testing NAD+ repletion therapy in cachectic mice reveals that NAD+ precursor, vitamin B3 niacin, efficiently corrects tissue NAD+ levels, improves mitochondrial metabolism and ameliorates cancer- and chemotherapy-induced cachexia. In a clinical setting, we show that muscle NRK2 is downregulated in cancer patients. The low expression of NRK2 correlates with metabolic abnormalities underscoring the significance of NAD+ in the pathophysiology of human cancer cachexia. Overall, our results propose NAD+ metabolism as a therapy target for cachectic cancer patients.

The patient population's characteristics reported in Table 1 includes the sex of the partecipants. The cohort studied includes both sexes and is balanced (64 females, 66 males, divided in 3 subgroups). Sex was determined based on self-reporting. The analysis was performed in an aggregated form in order to obtain a suffient numerosity and a consequent statistical significance of the results. In a follow-up study, given the currently ongoing enrollment, a sex-based analysis will bè performed.
The only relevant covariate characteristic was the age of the control group differing as compared to the pre-cachectic group (62.7 ± 12.9 vs 68.0 ± 13.3; P= 0.04). The cachectic group did not differ from either the control nor the pre-cachectic group. Information on drug use and co-morbidities was collected and did not show any difference among the groups.
From 2015 to 2020 consecutive patients with colorectal or pancreatic cancer and control patients undergoing surgery for benign diseases were enrolled at the 3rd Surgical Clinic of the University Hospital of Padova. No selection bias is present, since the sequentiality of medical access was the only criterion.
The research project was approved by the Ethical Committee for Clinical Experimentation of Padova (protocol number 3674/ AO/15).
Sample sizes were calculated using size power analysis methods for a prior determination, on the basis of the SD and effect size previously obtained using the experimental methods used in the study. With a type I error of 0.05 and a power of 0.80, we calculated the minimal sample size for each group to be at least six mice. Considering a likely drop-off effect of 10-20%, we set sample size for each group to eight mice. To reduce the SD, we minimized physiological variation using mice of the same sex and same age.
Outliers were identified using ROUT (C1=1%) and excluded from the analysis.
For ethical reasons (3Rs principles of animal experimentation), animal experiments were not repeated, unless performed with a different outcome/endpoint. The survival experiment in C26-F mice confirmed that niacin improves macroscopically animal health, even if the survival, likely depending on tumor progression, did not.
In the animal studies, all the mice were randomized and allocated to either the vehicle or the niacin treatment. In the human study, being observational, the allocation was based on internationally accepted diagnostic criteria.
The investigators were not blinded given their involvement in both group allocation and data collection / analysis. In an ideal condition, blinding is desirable in every research experiment. Unfortunately, the current study was performed in three independent small research units, therefore blinding would have required the inclusion of people with inedaquate skills with potential technical errors that would have negatively impacted on the quality of the results. Moreover, the experimental work performed in the three independent labs producing consistent results partially supports the fairness of the analyses. Puromycin (EQ0001, Kerafast): C2C12 myoblasts were starved of serum and leucine for 2 hr and then IGF-1 and leucine were added to the medium of some of the cells for 45 min. Puromycin (1uM) was added to the medium of some of the cells (lanes 3-6) 30 min before harvest. Puromycin incorporation was analyzied by western blot. TOMM20 (ab186735, Abcam): western blot analysis on PC-12 (rat) and NIH/3T3 (mouse) cell lines.
C26 carcinoma cells were a gift from Prof. Mario P. Colombo (Istituto di Ricovero e Cura a Carattere Scientifico National Cancer Institute, Milano, Italy) and used to induce experimental cachexia. The in vivo use of these cells is well established (doi: 10.1016/j.semcdb.2015.09.002.) and adoped in our lab in the last 15 years. C26 cells are not commercially available. Prof. MP Colombo obtained the cells from the laboratory that originated the clone in 1975 (PMID: 1149045) No authentication method was used.
C26 cells tested negative for mycoplasma contamination.
Not used.